The 2-chloroethylnitrosoureas including BCNU, CCNU, trans-4-Methyl CCNU and chlorozotocin represent an important class of antitumor drugs. These agents are all capable of causing cellular alkylation and, to a more variable degree, cellular carbamylation. Recent evidence points heavily towards alkylation events being most important for antineoplastic activity. Macromolecular alkylation events occur as 2-chloroethyl alkylations mostly upon nucleic acids rather than proteins. It is proposed that a major class of products are DNA phosphotriesters. These products as well as base alkylation products are capable of a second alkylation thus completing bifunctional alkylations. Research is proposed to elucidate both the quantity and nature of these phosphotriesters. Synthetic DNAs will be alkylated by a variety of 2-haloethylnitrosoureas, degraded enzymically to the nuclease resistant dideoxynucleoside phosphotriesters and the triesters separated and quantitated by high performance liquid chromatography. Similar studies will be conducted with chromatin to establish the alkylation events of chromatin associated DNA. These studies will provide very useful information for studies on the alteration of DNA structure and biological function by the 2-haloethylnitrosourea antitumor drugs.